Increased microvascular permeability is a hallmark of inflammation and characterizes the initial events in ischemia-reperfusion. Previous research has focused on how to prevent the onset and the maintenance of the elevated permeability. We propose to investigate the mechanisms that inactivate hyperpermeability in the inflammatory phase of ischemia-reperfusion and return it to baseline levels in striated muscle. We hypothesize that after a hyperpermeability phase, the postischemic muscle begins a process to inactivate hyperpermeability and restore the physiological barrier properties of the microvascular wall. We further propose that Epac and Rap-1 serve as `Barrier Enhancing Factors' and participate in the hyperpermeability-inactivation process. Maintenance of microvascular barrier properties is mainly regulated through factors controlling proteins that form intercellular adhesions. Cell-cell adhesion, in turn is regulated in many cells in part through feed-back signaling between small GTP-binding proteins and junctional proteins. The Specific Aims designed to test the hypothesis are: Specific Aim 1: To investigate the timed inactivation of hyperpermeability after ischemia- reperfusion induced inflammation. Specific Aim 2: To determine whether activation of Epac/Rap-1 inactivates hyperpermeability in ischemia-reperfusion. Specific Aim 3: To test whether endothelial Epac/Rap-1 signaling is responsible for restoration of barrier integrity. We will apply microscopy, computer-assisted image analysis and molecular biology approaches in striated muscle (in vivo) and in endothelial cells to elucidate the role of these relevant cAMP-stimulated factors in the inactivating phase of hyperpermeability in postischemic muscle. We will use endothelial cells exposed to oxygenation and reoxygenation to explore the cellular mechanisms. The body response to inflammation and to ischemia reperfusion is highly complex and involves well orchestrated molecular mechanisms. Knowledge of the timing between hyperpermeability and its "physiological" inactivation in ischemia-reperfusion should provide a window of opportunity for interventions promoting inactivation of hyperpermeability to prevent excessive edema, compartment syndrome and tissue damage. Our results should be of clinical relevance to revascularization in elective and emergency vascular surgery. PUBLIC HEALTH RELEVANCE: Our results will advance current understanding of the molecular mechanisms involved in the inactivation of increased permeability. The emerging data will be clinically relevant in the settings of revascularization. The results could serve as a basis for new adjuvant therapeutic approaches to assist vascular surgeons in preventing damage and returning function to postischemic muscles. [unreadable] [unreadable] [unreadable]